Method for Detecting a Mutation in a Microsatellite Sequence

ABSTRACT

The invention relates to a method for detecting a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, comprising subjecting said DNA sample to a digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising:
         a pair of primers for amplifying said target fragment of the DNA sample including said microsatellite sequence;   a first MS oligonucleotide (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence; and   a second oligonucleotide reference (REF) hydrolysis probe, labeled with a second fluorophore, wherein said second oligonucleotide REF probe is complementary to a wild-type sequence of said target DNA fragment which does not include said microsatellite sequence. The invention also encompasses methods for the diagnosis and prognosis of cancer and a method for determining the efficacy of a cancer treatment.

Microsatellites (MS) are tandem repeats of short DNA sequences that areabundant throughout the human genome. Owing to their high mutationrates, microsatellite sequences have been widely used as polymorphicmarkers in population genetics and forensics. Microsatellite instability(MSI) is a hypermutator phenotype that occurs in tumors with impairedDNA mismatch repair (MMR) and is characterized by widespread lengthpolymorphisms of microsatellite repeats due to DNA polymerase slippageas well as by elevated frequency of single-nucleotide variants (SNVs).MSI is caused by inactivation of MMR genes (for example, MLH1, MSH2,MSH3, MSH6 and PMS2) through somatic mutations, with increased risk ofcancer for those with inherited germline mutations (that is, Lynchsyndrome). MSI also occurs by epigenetic inactivation of MMR genes (forexample hypermethylation of the MLH1 and MSH2 promoters associated withthe somatic BRAF V600E mutation or deletion in the 3′ end of Ep-CAM), ordownregulation of MMR genes by microRNAs. MSI events within codingregions can alter the reading frame, leading to truncatedfunctionally-impaired proteins (see also Cortes-Ciriano et al., NatCommun. 2017 Jun. 6; 8:15180 and Copija et al., Int J Mol Sci. 2017 Jan.6; 18(1). pii: E107).

The MSI phenotype has been largely used as a molecular diagnostic toolfor gastrointestinal, endometrial and colorectal tumors, where it hasimportant implications for disease prognosis and rational planning oftreatment (Boland and Goel, Gastroenterology 2010 June;138(6):2073-2087.e3, Copija et al., Int J Mol Sci. 2017 Jan. 6; 18(1).pii: E107). MSI positive tumors are known to display uniquehistopathological and clinical features including specific location,poor differentiation, high lymphocytic infiltration and better prognosisassociated with low frequency of distant metastasis (Boland and Goel,Gastroenterology 2010; 138(6):2073-2087.e3).

Recent analyses have also identified MSI across several additionalcancer types, such as urinary tract, ovarian, prostate, lung, head andneck, liver and glioblastomas, suggesting a potential broaderapplication of MSI screening in clinical practice (Hause et al., NatMed. 2016 November; 22(11):1342-1350, Cortes-Ciriano et al., Nat Commun.2017 Jun. 6; 8:15180).

Indeed, MSI has recently emerged as the first pan-tumor biomarker likelyto predict clinical benefit from immune-checkpoint blockade therapy (Leet al., N Engl J Med. 2015 Jun. 25; 372(26):2509-20; Le et al., Science2017 Jun. 8. pii: eaan6733). Remarkably, via an Accelerated Approvalprocess, the FDA has recently granted the use of anti-PD-1 blockadetherapy for the treatment of adult and pediatric patients withunresectable or metastatic MSI positive or MMR-deficient solid tumors.

Molecular diagnosis of MSI is currently done by examining PCR productsof a few informative microsatellite loci of DNA extracted from tumorsamples (Bacher et al., Disease Markers 2004, 237-250). Disadvantages ofthis method include the requirement of capillary electrophoresis fordetection of shifts in allele size and the limited sensitivity of thetechnique, which requires a minimum tumor cellularity of 20% to achievereliable and robust results (Shi and Washington, Am J Clin Pathol 2012137:847-859). Recently, next generation sequencing methods (NGS) havebeen used for higher sensitivity and better precision in MSI detection(Salipante et al., Clin Chem 2014 Jun. 30 60 (9), 1192-1199; Hause etal., Nat Med. 2016 November; 22(11):1342-1350, Cortes-Ciriano et al.,Nat Commun. 2017 Jun. 6; 8:15180). Although clearly an improvement overthe method currently used in clinics, the sensitivity of 1% obtained byNGS still remains above the sensitivity of PCR-based assays.

Thus the development of a sensitive MSI diagnostic method usable oncirculating tumor DNA obtained from liquid biopsies remains of highclinical and therapeutic importance.

SUMMARY OF THE INVENTION

The authors have designed a digital PCR diagnostic method for detectingmicrosatellite instability, which can be performed on a DNA samplecontaining a very low concentration of target DNA.

The authors have indeed demonstrated that the achieved limit ofdetection (i.e., the lowest concentration likely to be reliablydistinguished from the limit of blank and at which the detection isfeasible), is 250 fold lower than the minimum cellularity threshold(i.e. at least 20% cellularity) required to determine the MSI status bythe pentaplex assay currently applied in clinical practice (see Bacheret al 2004, Disease Markers 20:237-250, see also Shi and Washington, AmJ Clin Pathol 2012 137:847-859). According to the results as shownherein the present new MSI detection assay is both highly specific andsensitive, as sensitivity could reach values, at least in theory,approaching 0.1%. This innovative approach also offers several otheradvantages including the simplicity of blood tests and reduced time ofanalysis. Taken together, the MSI diagnostic method of the inventionpromises better diagnostic accuracy and the unprecedented use of a MSIbiomarker in liquid biopsies for diagnosis and monitoring of diseasetreatment and disease progression.

A similar technique has been previously used for the detection of BRAFstatus in colorectal cancer (see Bidshahri et al., The Journal ofMolecular Diagnostic 2016, 18(2):190-204). However, the use of such atechnique has never been envisioned for the detection of a mutatedmicrosatellite sequence. Indeed, due to the size and more particularlyto the extreme repetitive nature of the microsatellite sequence, itwould be expected that the probe covering the microsatellite (MS probe,see below) would slip over the repeat sequence such that efficient orreliable hybridization of the probe could not be obtained.

Dietmaier et al. (Laboratory Investigation, 2001) describe a techniquefor the detection of a microsatellite sequence by RT-PCR and byanalyzing the melting point, using hybridization probes of specificsequences of the targeted markers. The Light Cycler HybProbeshybridization probes used in this document are not capable ofdiscriminating WT and mutant microsatellite sequences. Thereforeadditional melting point analyses are required after real time PCRamplification for identification of mutated microsatellites. Besides,the probes of Dietmaier et al. are not regarded as hydrolysis probes andare not relevant in the context of a digital PCR reaction.

The method of the invention is based on a single reaction with twohydrolysis probes located within the same amplicon. The first one coversthe full WT microsatellite sequence (MS probe). The second one is areference probe (REF) located in a non-variable region, which does notinclude the microsatellite sequence (MS) locus and is used to quantifydroplets with amplifiable DNA. Therefore, wild-type (WT) sequences willdisplay a double positive fluorescence signal coming from thehybridization of both the REF and MS probes, while droplets containingmutated microsatellite alleles will present a shifted signal thatresults from the hybridization of the REF probe only.

The present invention therefore relates to a method for detecting amutation in a microsatellite sequence locus of a target fragment from aDNA sample, comprising a step of subjecting said DNA sample to a digitalpolymerase chain reaction (dPCR) in the presence of a PCR solutioncomprising:

-   -   a pair of primers suitable for amplifying said target fragment        of the DNA sample including said microsatellite sequence;    -   a first oligonucleotide microsatellite (MS) hydrolysis probe,        labeled with a first fluorophore, wherein said first MS        oligonucleotide probe is complementary to a wild-type sequence        including the microsatellite sequence;    -   a second oligonucleotide reference (REF) hydrolysis probe,        labeled with a second fluorophore, wherein said second        oligonucleotide REF probe is complementary to a wild-type        sequence of said target DNA fragment, which does not include        said microsatellite sequence.

Preferably, the digital PCR (dPCR) is digital droplet PCR (ddPCR).

The target fragment of the DNA sample can be constitutional genomic DNAor genomic tumor DNA or circulating DNA.

The microsatellite sequence locus can be selected from the groupcomprising BAT-25, BAT-26, BAT-34c4, BAT-40, NR21, NR24, MONO-27,D2S123, D5S346, D175250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3,GTF21P1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1 and TDRD1. Inaddition, microsatellite sequences located in regions of the genomefrequently amplified in cancer (e.g. chr8q region of the human genome)can be selected to increase sensitivity.

Typically, the DNA sample is selected from the group consisting of tumortissue, disseminated cells, feces, blood cells, blood plasma, serum,lymph nodes, urine, saliva, semen, stool, sputum, cerebrospinal fluid,tears, mucus, pancreatic juice, gastric juice, amniotic fluid,cerebrospinal fluid, serous fluids.

The present invention also relates to a method according to any of thepreceding claims further comprising a step of measuring the fluorescencesignals associated with the REF and MS probes, wherein: the maximalfluorescence intensity signal associated with both the REF and MS probesindicates the presence of a wild-type microsatellite sequence in thetarget DNA fragment, while a shift in the fluorescence intensity signalassociated with the MS probe indicates the presence of a mutation in themicrosatellite sequence of the target DNA fragment

The present invention also relates to a method for the diagnostic ofcancers, diseases associated with mutations in mismatch repair (MMR)genes or familial tumor predisposition in a subject, comprising thedetection of a mutation in a microsatellite sequence locus of a targetDNA from a DNA sample as described above, wherein the target fragmentoriginates from a tumor.

The present invention also relates to a method for the prognosis ofcancers comprising the detection of a mutation in a microsatellitesequence locus of a target fragment from a DNA sample as describedabove, wherein the target fragment originates from a tumor.

The present invention also relates to a method for predicting theefficacy of a treatment in a subject suffering from a cancer, comprisingthe detection of a mutation in a microsatellite sequence locus of atarget fragment from a DNA sample as described above, wherein the targetfragment is originating from a tumor and wherein the treatment ispreferably immunotherapy such as immune checkpoint therapy.

The present invention also relates to a method of treatment of a cancerin a subject in need thereof comprising:

-   -   the detection of a mutation in a microsatellite sequence locus        of a target fragment from a DNA sample as described above, and    -   the administration to the subject of an immunotherapy if a        mutation is identified in a microsatellite sequence locus of the        target fragment,        wherein the target fragment of the DNA sample originates from a        tumor.

The present invention also relates to a method for the monitoring of apatient diagnosed with a tumor associated with impaired DNA mismatchrepair (MMR), or having suffered from such tumor, comprising thedetection of a mutation in a microsatellite sequence locus of a targetfragment from a DNA sample as described above,

-   -   wherein the target fragment of the DNA sample originates from a        tumor.

Lastly, the present invention also encompasses a kit for identifying amutation in a microsatellite sequence region of a target fragment from aDNA sample comprising:

-   -   a pair of primers suitable for amplifying said target fragment        from the DNA sample including said microsatellites sequence;    -   a first oligonucleotide hydrolysis probe (MS), labeled with a        first fluorophore, wherein said first oligonucleotide hydrolysis        probe is complementary to a wild-type sequence including the        microsatellite sequence;    -   a second oligonucleotide hydrolysis probe (REF), labeled with a        second fluorophore, wherein said second oligonucleotide        hydrolysis probe is complementary to a wild-type sequence of        said amplified DNA fragment, which does not include said        microsatellite sequence;    -   a thermostable polymerase.

DETAILED DESCRIPTION

A—Definitions

The following definitions are intended to assist in providing a clearand consistent understanding of the scope and detail of the followingterms, as used to describe and define the present invention:

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one.”

As used herein a “tumor” or a “neoplasm” (both terms can be usedinterchangeably) is an abnormal new growth of cells. The cells in aneoplasm usually grow more rapidly than normal cells and will continueto grow if not treated. As they grow, neoplasms can impinge upon anddamage adjacent structures. The term neoplasm can refer to benign(usually curable) or malignant (cancerous) growths.

A benign tumor, or neoplasm, is usually localized, and does not spreadto other parts of the body. Most benign tumors respond well totreatment. However, if left untreated, some benign tumors can grow largeand lead to serious disease because of their size. Benign tumors canalso mimic malignant tumors, and so for this reason are sometimestreated. Malignant tumors are cancerous growths. They are oftenresistant to treatment, may spread to other parts of the body (i.e.metastasis) and they sometimes recur after they were removed.

The term “cancer” is used herein for a malignant tumor.

“Allele”, as used herein, refers to one of several alternative forms ofa gene or DNA sequence at a specific chromosomal location (locus). Ateach autosomal locus an individual possesses two alleles, one inheritedfrom the father and one from the mother.

“DNA polymorphism”, as used herein, refers to the existence of two ormore alleles for a given locus in the population. “Locus” or “geneticlocus”, as used herein, refers to a unique chromosomal location definingthe position of an individual gene or DNA sequence. “Locus-specificprimer”, as used herein, refers to a primer that specifically hybridizeswith a portion of the stated locus or its complementary strand, at leastfor one allele of the locus, and does not hybridize efficiently withother DNA sequences under the conditions used in the amplificationmethod.

“Microsatellite sequence Locus” or “microsatellite sequence” are usedinterchangeably and refer to a region of genomic DNA that containsshort, repetitive sequence elements of one (1) to seven (7), typicallyone (1) to five (5), notably one (1) to four (4) base pairs in length.Each sequence repeated at least once within a microsatellite locus isreferred to herein as a “repeat unit”. Each microsatellite locustypically includes at least seven repeat units, notably at least tenrepeat units, and preferably at least twenty repeat units.“Microsatellite Instability” (hereinafter, “MSI”), as used herein,refers to a form of genetic instability in which alleles of genomic DNAobtained from certain tissue, cells, or bodily fluids of a given subjectare mutated at a microsatellite locus.

Mutations at microsatellite locus commonly typically includedeletion(s), addition(s) or substitution of at least one repeat unit ata microsatellite locus. Typically, MSI results in a change in length ata microsatellite locus, due to addition(s) or most frequentlydeletion(s).

As used herein, a “primer/probe set” refers to a grouping of a pair ofoligonucleotide primers and two oligonucleotide probes that eachhybridizes to a specific target nucleotide sequence. Saidoligonucleotide set consists of: (a) a forward discriminatory primerthat hybridizes to a first location of a nucleic acid sequence; (b) areverse discriminatory primer that hybridizes to a second location ofthe nucleic acid sequence downstream of the first location and (c) twoprobes, which hybridizes to a target sequence between the primers. Inother words, a primer/probe set consists of a pair of specific oligosthat anneal to opposite strands of a nucleic acid sequence (typicallyincluding a microsatellite sequence locus) so as to form an ampliconspecific to the nucleic acid sequence during the PCR reaction, and twoprobes, preferably fluorescent, which hybridize to (i.e., which arecomplementary to) a specific target sequence of the amplicon.

An “amplicon” refers to a nucleic acid fragment formed as a product ofnatural or artificial amplification events or techniques. Typically, theamplicon is produced by Polymerase chain reaction (PCR). “Amplifying”,as used herein, refers to a process whereby multiple copies are made ofone particular locus of a nucleic acid (i.e. a target sequence asmentioned above), such as genomic DNA. Amplification is accomplishedusing PCR (Saiki et al., 1985 Science 230: 1350-1354).

A “target (DNA) fragment”, or a “target (DNA) region” usedinterchangeably herein relates to the fragment of the DNA sample that isamplified by a pair of primers of a primer/probe set. According to theinvention, such target fragment includes a MS locus. A “targetsequence”, or “target DNA sequence” used interchangeably refers to a DNAsequence which is complementary to the first or the secondoligonucleotide probe.

As used herein, “digital PCR” refers to an assay that provides anend-point measurement that provides the ability to quantify nucleicacids without the use of standard curves, as is used in real-time PCR(see Sykes et al., 1992 Quantitation of targets for PCR by use oflimiting dilution. BioTechniques 13, 444-449, Vogelstein and Kinzler1999 Digital PCR. Proc Natl Acad Sci USA, 96:9236-9241 and Pohl andShihle 2004 Principle and applications of digital PCR. Expert Rev MolDiagn, 4:41-47, see also Monya Baker 2012 Nature Methods 9, 541-544).

In a typical digital PCR experiment, a PCR solution is made similarly toa classical TaqMan probe assay, which typically comprises the DNAsample, fluorescence-quencher probes (i.e., hydrolysis probes), primers,and a PCR master mix, which generally contains DNA polymerase, dNTPs,MgCl₂, and reaction buffers at optimal concentrations. The PCR solutionis then randomly distributed into discrete (i.e. individual) partitionsor compartments, such that some contain no target DNA and others containone or more target DNA copies, most preferably one target DNA copy.Thus, in these conditions, the reference signal associated with thepresence of the target DNA in the DNA sample in a given partition orcompartment should be theoretically 0 or 1. Obviously due to biologicalvariability for a population of partition or compartment, clouds areobserved corresponding respectively to the theoretic values 0 or 1.

The partitions are individually amplified to the terminal plateau phaseof PCR (or end-point) and then read for fluorescence, to determine thefraction of positive partitions.

If the partitions are of uniform volume, the number of target DNAmolecules present may be calculated from the fraction of positiveend-point reactions using Poisson statistics, according to the followingequation:

λ=−In(1−p)  (1)

wherein λ is the average number of target DNA molecules per replicatereaction and p is the fraction of positive end-point reactions. From λ,together with the volume of each replicate PCR and the total number ofreplicates analyzed, an estimate of the absolute target DNAconcentration is calculated.

Micro well plates, capillaries, oil emulsion, and arrays of miniaturizedchambers with nucleic acid binding surfaces can be used to partition thesamples in distinct compartments or droplets. Thus digital PCR as usedherein includes a variety of formats, including droplet digital PCR(ddPCR), BEAMing (beads, emulsion, amplification, and magnetic), andmicrofluidic chips.

“Droplet digital FOR” (ddPCR) refers to a digital PCR assay thatmeasures absolute quantities by counting nucleic acid moleculesencapsulated in discrete, volumetrically defined, water-in-oil dropletpartitions that support PCR amplification (Hinson et al., 2011, Anal.Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-1011). Asingle ddPCR reaction may be comprised of at least 20,000 partitioneddroplets per well.

A “droplet” refers to an individual partition of the PCR solution in adroplet digital PCR assay. In the following of the present application,digital PCR will be described in reference to droplet digital (ordigital droplet PCR, used interchangeably), however, as mentionedpreviously individual partition of the PCR solution according to theprinciple of digital PCR can be obtained according to a variety oftechniques. Therefore, the method of the invention as described below inreference to droplet digital PCR is not limited to this digital PCRtechnique and may be applied in a similar fashion to other digital PCRtechniques.

Techniques available for digital PCR include PCR amplification on amicrofluidic chip (Warren et al., 2006 Transcription factor profiling inindividual hematopoietic progenitors by digital RT-PCR. Proc Natl AcadSci USA 103, 17807-17812; Ottesen et al., 2006 Microfluidic digital PCRenables multigene analysis of individual environmental bacteria. Science314, 1464-1467; Fan and Quake 2007 Detection of aneuploidy with digitalpolymerase chain reaction. Anal Chem 79, 7576-7579). Other systemsinvolve separation onto microarrays (Morrison et al., 2006 Nanoliterhigh-throughput quantitative PCR. Nucleic Acids Res 34, e123) orspinning microfluidic discs (Sundberg et al., 2010 Spinning diskplatform for microfluidic digital polymerase chain reaction. Anal Chem82, 1546-1550) and droplet techniques based on oil-water emulsions(Hindson, Benjamin et al., 2011 High-Throughput Droplet Digital PCRSystem for Absolute Quantitation of DNA Copy Number. AnalyticalChemistry 83 (22): 8604-8610). Typically, digital PCR is selected fromdroplet digital PCR (ddPCR), BEAMing (beads, emulsion, amplification,and magnetic), and microfluidic chips. Preferably, the digital PCR isdroplet digital PCR.

A droplet supports PCR amplification of template molecule(s) usinghomogenous assay chemistries and workflows similar to those widely usedfor real-time PCR applications (Hinson et al., 2011, Anal. Chem.83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-1011). Oncedroplets are generated, they can be transferred on a PCR plate andemulsified PCR reactions can be run on a thermal cycler under aclassical program such as for example described in the Biorad'sGuideline for ddPCR(http://www.bio-rad.com/webroot/web/pdf/Isr/literature/Bulletin_6407.pdf).

Droplet digital PCR may be performed using any platform that performs adigital PCR assay that measures absolute quantities by counting nucleicacid molecules encapsulated in discrete, volumetrically defined,water-in-oil droplet partitions that support PCR amplification. Thestrategy for droplet digital PCR may be summarized as follows: The PCRsolution containing the DNA sample is diluted and partitioned intothousands to millions of separate reaction chambers (water-in-oildroplets) so that each contains one or no copies of the nucleic acidmolecule of interest.

The number of “positive” droplets detected, which contain the targetamplicon (i.e., target DNA fragment) (i.e., according to the presentinvention REF positive droplets), versus the number of “negative”droplets, which do not contain the target amplicon (i.e., REF negativedroplets), may be used to determine the number of copies of the nucleicacid molecule of interest that were in the original sample.

Examples of droplet digital PCR systems include the QX100™ DropletDigital PCR System by Bio-Rad, which partitions samples containingnucleic acid template into 20,000 nanoliter-sized droplets; and theRainDrop™ digital PCR system by RainDance, which partitions samplescontaining nucleic acid template into 1,000,000 to 10,000,000picoliter-sized droplets.

The benefits of dPCR and more particularly ddPCR technology include:

-   -   Absolute quantification, as ddPCR technology provides an        absolute count of target DNA copies per input sample without the        need for running standard curves.    -   Unparalleled precision, as the massive sample partitioning        afforded by ddPCR enables the reliable measurement of small fold        differences in target DNA sequence copy numbers among samples.    -   Increased signal-to-noise ratio: high-copy templates and        background are diluted, effectively enriching template        concentration in target-positive partitions, allowing for the        sensitive detection of rare targets.    -   Removal of PCR bias, as error rates are reduced by removing the        amplification efficiency reliance of qPCR, enabling the        detection of small (1.2-fold) differences.    -   Simplified quantification, since neither calibration standards        nor a reference required for absolute quantification.    -   Reduced consumable costs, as reaction volumes are in the pico-        to nanoliter ranges, reducing reagent use and the sample        quantity required for each data point.    -   Lower equipment costs, as the emulsion-based reaction system        means that the PCR reactions can be performed in a standard        thermal cycler without complex chips or microfluidics.    -   Superior partitioning, since ddPCR technology yields 20,000        droplets per 20 μl sample, nearly two million partitioned PCR        reactions in a 96-well plate, whereas chip-based digital PCR        systems produce only hundreds or thousands of partitions. The        greater number of partitions also yields higher accuracy.

The term “melting temperature” or “Tm” refers to the temperature atwhich a polynucleotide dissociates from its complementary sequence.Generally the Tm may be defined as the temperature at which one-half ofthe Watson-Crick base pairs in a duplex nucleic acid molecule are brokenor dissociated (i.e. are “melted”) while the other half of theWatson-Crick base pair remain intact in a double stranded conformation.In other words, the Tm is defined as the temperature at which 50% of thenucleotides of two complementary sequences are annealed (double strands)and 50% of the nucleotides are denatures (single strands). The Tm can beestimated by a number of methods, such as for example by anearest-neighbor calculation as per Wetmur 1991 (Wetmur 1991 DNA probes:applications of the principles of nucleic acid hybridization. Crit RevBiochem Mol Biol 26: 227-259, hereby incorporated by reference) or bycommercial programs including Oligo™ Primer Design and programsavailable on the internet. Alternatively, the Tm can be determinedthrough actual experimentation. For example, double-stranded DNA bindingor intercalating dyes, such as ethidium bromide or SYBR-green (MolecularProbes) can be used in a melting curve assay to determine the actual Tmof the nucleic acid.

As used herein, the term “critical denaturation temperature” or “Tc”refers to a temperature below the Tm of the wild type sequence, at whichtemperature a duplex of the wild-type sequence and the mutant sequencewill melt. (In some instances, this temperature may be one at which ahomoduplex of the mutant sequences also melts). The critical denaturingtemperature (Tc) is the temperature below which PCR efficiency dropsabruptly for a given nucleic acid sequence.

B—Method for Identifying a Mutation in a Microsatellite Sequence Locusof a DNA Sample

The present invention relates to a method for the detection of amutation in a target microsatellite sequence (MS) locus of targetfragment from a DNA sample, comprising a step of subjecting said DNAsample to a polymerase chain reaction (PCR) in the presence of:

-   -   a pair of primers suitable for amplifying said target fragment        of the DNA sample including the said MS locus;    -   a first MS oligonucleotide probe, labeled with a first        fluorophore, wherein said first MS oligonucleotide probe is        complementary to a first wild-type target sequence including the        microsatellite sequence;    -   a second oligonucleotide reference (REF) probe, labeled with a        second fluorophore, wherein said second oligonucleotide REF        probe is complementary to a second wild-type target sequence of        said amplified DNA fragment which does not include the said        microsatellite sequence.

The DNA from the DNA sample and in particular the target DNA (or targetDNA fragment), can be genomic DNA or DNA issued from reverse RNAtranscription. The genomic DNA may be constitutional DNA, DNAoriginating from a tumor (i.e. tumor genomic DNA), notably a malignanttumor. Typically also, the target DNA fragment is cell-free DNA, such ascirculating DNA. In particular, the target DNA fragment can be cell-freetumor DNA, notably circulating tumor DNA, or cell-free fetal DNA (i.e.,fetal DNA circulating in the maternal blood stream).

The method uses a primer/probe set as previously defined.

Preferably, the primer pair is typically designed so as to have a Tmlower than the Tc of the reaction. The pair of primer can be designedusing available computer programs. Typically, the probes according tothe invention are hydrolysis probes (also named TaqMan probes).Hydrolysis probes have a fluorophore covalently attached to their 5′-endof the oligonucleotide probe and a quencher.

Oligonucleotide probes are detectably labeled with a fluorescent labelwhich can be selected, for example, from the group consisting of FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein, FITC, IRD-700/800, CY3,CY5, CY3.5, CY5.5, HEX, TET (5-tetrachloro-fluorescein), TAMRA, JOE,ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, TexasRed, Yakima Yellow, Alexa Fluor PET, Biosearch Blue™, Marina Blue®,Bothell Blue®, Alexa Fluor®, 350 FAM™, SYBR® Green 1, Fluorescein,EvaGreen™, Alexa Fluor® 488 JOE™, 25 VIC™, HEX™, TET™, CAL Fluor®Gold540, Yakima Yellow®, ROX™, CAL Fluor® Red 610, Cy3.5™, Texas Red®, AlexaFluor® 568 Cry5™, Quasar™ 670, LightCycler Red640®, Alexa Fluor 633Quasar™ 705, LightCycler Red705®, Alexa Fluor® 680, SYT0®9, LC Green®,LC Green® Plus+, and EvaGreen™ Preferably, the detectable label isselected from 6-carboxyfluorescein, FAM, or tetrachlorofluorescein,(acronym: TET), Texas Red, Cyanin 5, Cyanine 3, or VIC™ The quencher maybe an internal quencher or a quencher located in the 3′ end of theprobe. Typical quenchers are tetramethylrhodamine, TAMRA, Black HoleQuencher or nonfluorescent quencher. Hydrolysis probes usable accordingto the invention are well-known in the field (see notablyhttp://www.sigmaaldrich.com/technical-documents/articles/biology/quantitative-per-and-digital-per-detection-methods.html).The quencher molecule quenches the fluorescence emitted by thefluorophore when excited by the cycler's light source typically via FRET(Forster Resonance Energy Transfer). As long as the fluorophore and thequencher are in proximity, quenching inhibits any fluorescence signals.Such probes are designed such that they anneal within the target regionamplified by a specific set of primers. As the Taq polymerase extendsthe primer and synthesizes the nascent strand, the 5′-3′ exonucleaseactivity inherent in the Taq DNA polymerase then separates the 5′reporter from the 3′ quencher, which provides a fluorescent signal thatis proportional to the amplicon yield.

The first and second probes according to the invention are locatedwithin the same amplicon. The probes are designed according to thewell-established practice in the art to preferably minimize PCR artifactand to specifically hybridize with the sequences as defined below. Thefirst and second probes are labeled with distinct fluorophores in orderto allow separate detection of their respective signal.

In some embodiments, the hydrolysis probes according to the inventioninclude a minor groove binder (MGB) moiety at their 3′ end. Such MGBtypically increases the melting temperature (Tm) of the probe andstabilizes probe—target hybrids.

The oligonucleotide probes have a nucleotide sequence length of about 10to about 50.

Preferably, the oligonucleotide probes (and in particular the MS probe)have a nucleotide sequence length of about 15 to 40, or 25 to 50 ornotably 15 to 35.

Preferably, the oligonucleotide probes (and in particular the MS probe)have a nucleotide sequence length of about 20 to 40, or 30 to 50 ornotably 30 to 40.

The first probe according to the invention (also named MS probe)hybridizes with a first wild-type target sequence of the amplifiedtarget DNA fragment, which includes a microsatellite sequence locus.Preferably the probe covers the full wild-type microsatellite sequenceand extends further a few nucleotides on each extremity (typically 1 to10 nucleotides, notably 2 to 8, preferably 2 to 6, most preferably 2 to5 or 2 to 4) to confer both its ability to bind properly and theresulting destabilization in case of microsatellite instability. Inother words, the probe size is designed to confer its ability to bindproperly to the wild-type microsatellite sequence, while preventinghybridization of the MS probes in the presence of a mutation in themicrosatellite sequence.

The second probe of the invention (also named REF probe) hybridizes witha second wild-type target sequence of said amplified DNA fragment, whichdoes not include the said microsatellite sequence. In particular, saidsecond probe may partially overlap with the said microsatellite sequenceor be located outside of the said microsatellite sequence.

Preferably the second probe according to the invention is locatedoutside of the said microsatellite sequence.

Various microsatellite sequence loci can be targeted in the firstwild-type target sequence according to the invention. Microsatellitesequence loci or markers than can be targeted according to the inventionare notably described in Bacher et al., 2004 Disease Markers 20,237-250, as well as in Hause et al., 2016 Nat Medicine November22(11):1342-1350). Preferably, targeted microsatellite sequence loci (ormicrosatellite markers) are selected from microsatellites found to behighly associated with MSI positive tumors, based on their frequency ofinstability in colon, endometrial, rectal and stomach adenocarcinomas.

Preferably, targeted microsatellite sequence loci are located in regionsfrequently amplified in tumors.

For example, a targeted microsatellite sequence locus can be selectedfrom BAT-25, BAT-26, BAT-34c4, BAT-40, NR21, NR24, MONO-27, D2S123,D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3, GTF21P1,LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1 and TDRD1.

In one embodiment, the target microsatellite sequence locus may also beselected among the Bethesda panel, which comprises BAT-25, BAT-26,D2S123, D5S346 and D17S250.

Mononucleotide repeat loci have been shown to be very susceptible toalteration in tumors with dysfunctional DNA mismatch repair systems(Parsons, 1995 supra), making such loci particularly useful for thedetection of cancer and other diseases associated with dysfunctional DNAmismatch repair systems, such that mononucleotides MSI markers may bepreferred.

In one embodiment of the invention, a targeted microsatellite sequencelocus is BAT-26 and/or ACVR2A and/or DEFB105A and DEFB105B.

More generally, appropriate microsatellite sequence loci that can betargeted according to the invention are short microsatellite sequences(typically comprising 8 to 30, notably 8 to 25, preferably, 8 to 20,most preferably 8 to 15 or 8 to 12 nucleotides) such as the targetmicrosatellite sequence locus exemplified in the group consisting ofD2S123, D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3,GTF21P1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1 and TDRD1.

Depending on the microsatellite locus, probes of various sizes and G/Ccontent may also be used. For example, probes of more than 30nucleotides and/or with a G/C content of less than 30% may be used. Thisis notably the case with BAT-26. As a matter of example an MS probe ofSEQ ID No.:4, which hybridize with a sequence including the BAT-26microsatellite sequence can be used. Primers of sequences SEQ ID No: 1-2as well as REF probe and MS probe of respectively SEQ ID No: 3 and 4represent an illustrative set of primer/probe that can be used accordingto the invention.

According to the present invention, amplification of the target DNAfragment occurs with a digital PCR technique. Typically, in such atechnique, the PCR solution is divided in multiple compartments ordroplets, which are made to run PCR individually. Typically also most ofthe compartments or droplets contain either 0 or 1 copy of the targetDNA fragment which is to be amplified.

To circumvent the technical challenges associated to the amplificationof low complexity sequence such as the microsatellite sequence, a seriesof modifications may be provided to Biorad's Guideline for ddPCR asmentioned above, in order to achieve proper hybridization of the MSprobe to WT alleles. Reaction annealing temperature and/or extensiontime may be increased. Typical annealing temperature according toBiorad's Guidelines is 55° C. Said annealing temperature may beadvantageously increased from 3 to 15° C.

Thermal cycling is performed to endpoint. Thus after multiple PCRamplification cycles (i.e. after completing PCR cycles), the raw PCRdata are then collected by measuring the fluorescence signal associatedwith both the REF and MS probes for each droplet. Droplets containing WTtarget fragments display a double positive fluorescence signal comingfrom the hybridization of both the REF and MS probes (REF+/MS+droplets). The non-hybridization (or inefficient hybridization) of theMS probe in droplets containing mutated microsatellite alleles leads toa shift of the droplet cloud on the 2D graph, toward a single REFpositive (REF+) population, which is proportional to the fraction ofdroplets containing mutant microsatellite alleles.

Typically, raw dPCR (or ddPCR) data are collected after PCR cycling byreading or measuring the fluorescence signal associated with the REF andMS probes for each droplet.

The PCR data collection step is typically performed in an opticaldetector (for example the Bio-Rad QX-100 droplet reader can be used inddPCR). Preferably at least a two-color detection system is used (forexample to detect FAM and either HEX or VIC fluorescent labels).Droplets clouds can typically be established on 2D graphs by plottingthe fluorescence level for each probe per droplet. In some embodiments,analysis may be achieved with appropriate software (such as theQuantaSoft v1.7.4 software for ddPCR or the ddPCR package on R[https://cran.r-project.org/web/packages/ddper/index.html]. Quantasoftallows manual assignment of the droplets to the single REF positive orthe double REF/MS positive population (i.e. or clouds). The R packagedefines thresholds in an automatic way to avoid bias that might beintroduced by manual assignment.

The number of droplets that are positive for the reference probe (REFprobe) can be used to quantify the total number of target DNA fragmentsin the sample. The fraction of positive droplets can then be fitted to aPoisson distribution to determine the absolute initial copy number ofthe target DNA fragment in the input reaction mixture in units ofcopies/μl.

In droplets containing a wild-type target DNA (no mutation in thetargeted MS sequence), a maximum fluorescence signal is observed forboth the REF and the MS probes. At the contrary, in droplets containinga mutated sequence in the amplified target DNA fragment (i.e. a mutationin the microsatellite sequence), a shift in the fluorescence intensityis observed for the signal associated with the MS probe.

Most preferably, the digital PCR reaction is designed to ensure thatmost droplets contain either 0 or 1 copy of targeted DNA fragment(notably depending on the quantity of DNA loaded in the reaction. Inthese conditions, an optimal separation of the WT (REF+/MS+ signals) vs.mutated microsatellite (or MSI) clouds (single REF+ signal) can beobserved. It must be noted that due to biological variability thatdroplets classified in the single REF+ signal may include a residual(i.e., non-significant) MS signal. A threshold, under which a MS signalis considered as “a residual MS signal” can be determined by the oneskilled in the art according to classical signal analysis techniques.Said threshold can be typically set using the R package as mentionedpreviously.

Typically mutant allele frequency can be determined from droplet countsthrough manual assignment of WT and mutated microsatellite dropletclouds. As mentioned above, identification of a droplet population witha single signal from the REF probe indicates the presence of a mutatedmicrosatellite sequence in the DNA sample.

Mutant allele frequency which can be determined as mentioned above canbe compared with a control mutated allele frequency obtained from acontrol DNA sample. The control DNA sample may be a wild-type sample ora sample or cell line collected in a subject, diagnosed with a MSIpositive tumor or with a disease associated with a mutation in the DNAmismatch repair, at a prior time point, during the time-course of thedisease and/or during the time course of the treatment.

As used herein, the term “sample” refers to anything which may containDNA and notably the DNA fragment to be amplified. In some embodiment,the “sample” contains RNA and is therefore submitted to a reversetranscription step. The sample may be a biological sample, such as abiological fluid or a biological tissue. Examples of biological fluidsinclude urine, blood, plasma, serum, saliva, semen, stool, sputum,cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juiceamniotic fluid, serous fluids such as pericardial fluid, pleural fluidor peritoneal fluid.

Biological tissues are aggregate of cells, usually of a particular kindtogether with their intercellular substance that form one of thestructural materials of a human, animal, plant, bacterial, fungal orviral structure, including connective, epithelium, muscle and nervetissues. Examples of biological tissues also include organs, tumortissue, lymph nodes, arteries and disseminated cell(s). The tissue canbe fresh, freshly frozen, or fixed, such as formalin-fixedparaffin-embedded (FFPE) tissues. The sample can be obtained by anymeans, for example via a surgical procedure, such as a biopsy, or by aless invasive method, including, but not limited to, abrasion or fineneedle aspiration. Preferably, the DNA sample is selected from the groupconsisting of: tumor tissue, disseminated cells, feces, blood cells,blood plasma, serum, lymph nodes, urine, saliva, semen, stool, sputum,cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice,amniotic fluid, cerebrospinal fluid, serous fluids such as pericardialfluid, pleural fluid or peritoneal fluid.

The DNA and notably the target DNA fragment can be genomic DNA or DNAissued from reverse transcriptase. The genomic DNA can be constitutionalDNA, tumor DNA or fetal DNA. In some embodiments, notably when thesample is a biological fluid, the DNA sample may contain cell-free DNA(cfDNA), or circulating DNA. Early studies have shown that tumor DNA isreleased into the circulation, and is present in particularly highconcentrations in plasma and serum in a number of different types ofcancer (Leon et al., 1977 Cancer Res 37:646-650; Stroun et al., 1989Oncology 46:318-322). Thus, DNA sample according to the invention cancontain cell-free tumor DNA or circulating tumor DNA. In anotherembodiment, the DNA sample contains cell-free fetal DNA. Due to its highsensitivity, the method of the invention can be used on plasma samplecontaining low concentration of circulating, or cell-free target DNAsuch as cell-free or circulating tumor DNA or fetal DNA. In someembodiments of the present invention, the DNA can be obtained fromreverse transcription of an RNA sample.

Typically a DNA sample according to the invention is obtained from asubject. The subject, or the patient (both terms can be usedinterchangeably) of the invention is a mammal, typically a primate, suchas a human. In some embodiments, the primate is a monkey or an ape. Thesubject can be male or female and can be any suitable age, includinginfant, juvenile, adolescent, adult, and geriatric subjects. In someembodiments, the subject is a non-primate mammal, such as a rodent.

In some embodiments of the invention, the subject has a cancer, is inremission of a cancer, or is at risk of suffering from a cancer notablybased on family history. In some embodiments for example the subject hasfamilial tumor predisposition.

In some embodiment, the subject is suffering from, is in remission, orhas familial cancer predisposition, notably the subject is sufferingfrom or is at risk of suffering from a disease caused by mutations inmismatch repair (MMR) genes, such as Constitutional mismatch repairdeficiency syndrome (CMMRD syndrome) or Lynch syndrome.

The cancer may be a solid cancer or a “liquid tumor” such as cancersaffecting the blood, bone marrow and lymphoid system, also known astumors of the hematopoietic and lymphoid tissues, which notably includeleukemia and lymphoma. Liquid tumors include for example acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), acutelymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL),(including various lymphomas such as mantle cell lymphoma ornon-Hodgkins lymphoma (NHL). Solid cancers notably include cancersaffecting one of the organs selected from the group consisting of colon,rectum, skin, endometrium, lung (including non-small cell lungcarcinoma), uterus, bones (such as Osteosarcoma, Chondrosarcomas,Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, andChordomas), liver, kidney, esophagus, stomach, bladder, pancreas,cervix, brain (such as Meningiomas, Glioblastomas, Lower-GradeAstrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, andMetastatic brain cancers), ovary, breast, head and neck region, testis,prostate and the thyroid gland.

In some embodiments of the present invention the cancer is theConstitutional mismatch repair deficiency syndrome (CMMRD syndrome) orthe Lynch syndrome

In the context of the present invention, a cancer (or a tumor)associated with MSI is also named a MSI positive cancer (or a MSIpositive tumor) and relates to a cancer (or tumor) wherein the genomictumor DNA exhibits at least one mutation in a microsatellite sequence. AMSI positive cancer may thus be any of the cancers as listed abovewherein the genomic tumor DNA exhibits at least one mutation in amicrosatellite sequence

Clinical Applications: Diagnostic and Prognosis Methods, TherapeuticTreatment and Patient Monitoring.

The method for identifying a mutated microsatellite sequence in a targetDNA fragment as described above has several major and direct clinicalapplications.

First, as previously mentioned, microsatellite instability is ahypermutator phenotype that occurs in tumors associated with impairedDNA mismatch repair (MMR). MSI has thus been associated with a greatvariety of cancers such as but not limited to colorectal cancers,gastric cancer, endometrium cancer, ovarian cancer, urinary tractcancer, brain cancer, and breast cancer. MSI is most prevalent as theconsequence of colon cancers. MSI is typically found the Constitutionalmismatch repair deficiency syndrome (CMMRD syndrome) or the Lynchsyndrome.

Therefore, detection of a mutated microsatellite sequence according tothe method as previously described can be used in the diagnostic ofcancers as previously defined, in particular of cancers (as previouslydefined), which are associated with impaired DNA mismatch repair andnotably of MSI positive cancers (or tumors).

In one embodiment of the invention, detection of a mutatedmicrosatellite sequence according to the method can also be used in thediagnostic of diseases which are caused by mutations in mismatch repair(MMR) genes, such MSI positive tumors notably such as Constitutionalmismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome,or in the diagnostic of familial tumor predisposition in a subject.

Thus in one aspect, the present invention relates to a method for thediagnostic of cancers, notably of diseases associated with mutations inmismatch repair (MMR) genes, such as MSI positive tumors, and/or offamilial tumor predisposition to cancer in a subject, comprising thedetection of a mutation in a microsatellite sequence locus of a targetDNA from a DNA sample according to the present invention. Typically thetarget DNA is genomic DNA originating from a tumor. The sample can beobtained from a subject as previously described. In one embodiment,detection of a mutated microsatellite sequence in a DNA sample from asubject indicates that the subject is suffering from a disease caused bymutations in the MMR genes such as MSI positive tumors, notably CMMRD orLynch syndrome. Detection of a mutated microsatellite sequence in a DNAsample from a subject may also indicate that the subject has familialtumor predisposition such as in the CMMRD or Lynch syndrome.

Mutations in MMR genes include addition, deletion or substitution, inparticular single nucleotide variations (SNVs), as well as epimutations(such as DNA hypermethylation).

The prevalence of MSI positive tumors is higher in colorectal cancers,gastric cancers, endometrium cancers. However, MSI has been found at alower prevalence in virtually all type of cancers (see Hause et al.,2016 Nature Medicine), albeit with low prevalence. As previouslymentioned, the MSI phenotype of the cancer (i.e. positive or negative)has important implications in cancer prognosis and rational planning oftreatment (Boland and Goel, Gastroenterology 2010). Therefore even inthe case of cancers with low MSI positive prevalence, it remains of highrelevance to identify whether the patient is suffering from a MSIpositive tumor or a MSI negative tumor. The method of the presentinvention can therefore be used in the prognosis of various cancers.Identification of a positive MSI cancer is generally associated with abetter prognosis.

Thus, the present invention also relates to a method for the prognosisof cancers (as previously defined) comprising the detection of amutation in a microsatellite sequence locus of a DNA sample according tothe present invention. In some embodiment, identification of a mutatedmicrosatellite sequence in the sample, preferably a DNA sampleoriginating from a tumor, indicates that said tumor is MSI positive.

In the therapeutic contexts as above mentioned, the methods of theinvention are particularly useful as its great sensitivity allowsdetection of microsatellite instability in DNA samples containing verylow concentrations of target DNA. The method of the invention cantherefore be routinely performed on biological samples such as bloodsamples, plasma samples, urine or even feces. Typically, the methods ofthe invention are performed on blood sample or plasma sample and thetarget DNA is a cell-free DNA, such as a circulating tumor DNA. Thispoint is particularly relevant for diseases such as CMMRD, which involvecerebral tumor with no biopsy access.

The present invention also relates to a method for predicting theefficacy of a treatment, as reports have shown for example thatcolorectal cancer patients with MMR deficiency have better responses toimmunotherapy by PD-1 immune checkpoint blockade and show improvedprogression-free survival. Therefore, identification of patientssuffering from cancer associated with MSI (i.e. MSI positive cancer ortumor) is of high clinical relevance for selection of an appropriatetherapeutic strategy.

Thus, another aspect of the present invention concerns a method forpredicting the efficacy of a treatment in a subject suffering from acancer, wherein said method comprises the detection of a mutation in amicrosatellite sequence locus of a target DNA fragment from a subjectDNA sample as previously described. Preferably, the target DNA fragmentis originating from a tumor. Typically the DNA sample is obtained from asubject suffering from a tumor and/or having familial cancerpredisposition.

The present invention also proposes a method of treatment of a cancer ina subject in need thereof comprising the detection of a mutation in amicrosatellite sequence locus of a target DNA fragment from a DNA sampleaccording to the methods as herein described. Typically the target DNAfragment is originating from a tumor. Typically also the DNA sample isobtained from a subject suffering from a tumor and/or having familialcancer predisposition.

Preferably, the treatment is immunotherapy. Immunotherapy includes butis not limited to immune checkpoint modulators (i.e. inhibitors and/oragonists), monoclonal antibodies, cancer vaccines.

Most preferably, the treatment comprises administration of immunecheckpoint modulators such as anti-PD-1 and/or anti-PDL-1 inhibitors.

Preferably, immunotherapy is administered to the subject if a mutationin a microsatellite sequence locus of a target DNA (notably a targettumor DNA) from a DNA sample is detected.

Furthermore, the method of the invention for detecting microsatelliteinstability may also be used for the monitoring of a subject diagnosedwith a tumor associated with impaired DNA mismatch repair. Preferably,said monitoring is performed during the time course of the treatment.The method may also be used for the monitoring of cancer relapse in asubject having suffered from a tumor associated with impaired DNAmismatch repair. Thus, in another aspect, the present invention alsoprovides a method for the monitoring of a patient diagnosed with a tumorassociated with impaired DNA mismatch repair, or having suffered fromsuch a tumor, comprising the detection of a mutation in a microsatellitesequence locus of a target tumor DNA from a DNA sample selected from aplasma or a serum sample obtained from a subject diagnosed with a tumorassociated with impaired DNA mismatch repair or having suffered from atumor associated with impaired DNA mismatch repair. In patient havingsuffered from a tumor associated with impaired DNA mismatch repair,detection of microsatellite instability in circulating tumor DNA may beindicative of a relapse.

Multiplexed Assays for the Detection of a Mutation in a MicrosatelliteSequence Locus of a Target DNA from a DNA Sample

The power for detecting the presence of MSI in tissues associated with aparticular disease, such as cancerous tumors, can be increasedtremendously by multiplexing multiple markers. Thus, in the context ofthe invention more than one set of primer/probe as previously definedcan be used in a multiplexed assay such that more than onemicrosatellite sequence locus (i.e.; a panel microsatellite sequenceloci) as previously defined can be targeted.

As a matter of example, microsatellite sequence loci of the panel formultiplexed assays according to the invention can be selected among thegroup consisting of BAT-25, BAT-26, BAT-34c4, BAT-40, NR21, NR24,MONO-27, D2S123, D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43,DOCK3, GTF21P1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1 and TDRD1and in any of the groups as previously defined.

Such multiplexed assay is particularly useful in the clinicalapplications as previously described.

Preferably, in multiplexed assays, the primers pairs are designed usingavailable computer programs such that upon amplification the resultingamplicons are predicted to have the same melting temperature.

When in the digital range (where all compartments contain either 0 or 1target molecule) it is possible to multiplex qPCR assays without concernfor competition or cross reactivity, as each target-containing reactionwill proceed with the target binding to its primers/probe specifically,whereas no reaction will occur in compartments without targets. Havingeach molecule in a separate reaction compartment allows both high andlow abundance targets to be counted in the same experiment withoutconcern for “swamping out” the low abundance target (since eachcompartment has at most 1 target, independent of its concentration inthe average sample volume). When more than one target is counted (e.g.,in a duplex assay format), ratios of the counts for one target relativeto another (e.g., mutant allele vs. wild type allele) enable “absoluteratios” to be quantified, using one of the targets as an internalnormalizing reference (e.g., how many amplifiable genome equivalentswere loaded) that has gone through the identical experiment as the othertargets assayed.

In addition, since dPCR is performed as an endpoint reaction (PCR is runto completion before measuring fluorescence), having true single targetmolecules in isolation allows multiplexing based on probe intensity(Zhong, Bhattacharya, et al., 2011 Multiplex digital PCR: breaking theone target per color barrier of quantitative PCR. Lab Chip,11:2167-2174). By adding the target-specific fluorescent assay at alimiting concentration, a compartment with that target molecule will bePCR-positive, but with a limited brightness at PCR endpoint. To count asecond target type, a different target-specific probe with the same“color” (i.e. with the same fluorophore) is added at a differentconcentration. A compartment with the second target will have a brightersignal at PCR endpoint than a compartment with the first target,providing separate clouds and thus enabling separate counts for eachtarget. Thus, combinations of both different color probes and differentconcentration probes can be used to multiplex at higher levels.

Kits:

The present invention also encompasses kit for identifying a mutation ina microsatellite sequence region of a DNA sample comprising aprimer/probe set comprising:

-   -   a pair of primers suitable for amplifying a target DNA fragment        of said DNA sample including the said microsatellites sequence;    -   a first oligonucleotide probe, labeled with a first fluorophore,        wherein said first oligonucleotide probe is complementary to a        wild-type sequence including the microsatellites sequence;    -   a second oligonucleotide probe, labeled with a second        fluorophore, wherein said second oligonucleotide probe is        complementary to a wild-type sequence of said amplified DNA        fragment located outside of the said microsatellite sequence;    -   a thermostable DNA polymerase.

Thermostable DNA polymerases are typically described in Newton andGraham 1994 In: PCR, BIOS Scientific Publishers, Ltd., Oxford, UK. 13.Advantageously, the thermostable polymerase is the Taq polymerase.

In one aspect, the kit comprises more than one primer/probe set, whereinthe primer/probe sets allows amplification and detection of target DNAfragments comprising distinct microsatellite sequences.

The kit as above mentioned can be used in the clinical applications aspreviously described.

FIGURES

FIG. 1 : A-C. 2-D fluorescence amplitude scatter plots of BAT-26 ddPCRMSI assay using HCT-116 cell line DNA (MSI-H), PBMC (VVT) or a 10%dilution of HCT-116 in WT DNA. D-F. 2-D fluorescence amplitude scatterplots of DEFB105A/B ddPCR MSI assay using HCT-116 cell line DNA (MSI-H),PBMC (WT) or a 10% dilution of HCT-116 in WT DNA. G-I. 2-D fluorescenceamplitude scatter plots of ACVR2A ddPCR MSI assay using HCT-116 cellline DNA (MSI-H), PBMC (WT) or a 10% dilution of HCT-116 in WT DNA.Droplets containing WT alleles are positive for both FAM and VICsignals, while droplets containing MSI alleles are positives for VICsignal only.

FIG. 2 : Correlation curves obtained for BAT-26 (A), DEFB105A/B (B) andACVR2A (C) assays for observed versus expected MAFs in reconstitutedmutant serial dilutions (10%, 5%, 2.5%, 1.25, 0.63%, 0.31%, 0.16%,0.08%, 0.04%, 0.02%, 0.01%). Dotted lines: LOB, estimated as the upper95% CI of false-positive calls in at least 53 independent ddPCRreactions with WT DNA.

FIG. 3 : Correlation between ctDNA fractions estimated by BAT-26 (A),ACVR2A (B) or DEFB105A/B (C) ddPCR assays and a ddPCR assay targetingspecifically BRAF^(V600E) mutation.

FIG. 4 : 2-D fluorescence amplitude scatter plot illustratingfluorescence signals obtained with a triplex assay targetingsimultaneously BAT-26, ACVR2A and DEFB105A/B microsatellite markersusing a 10% dilution of HCT-116 cell line in WT DNA. Results obtainedwith annealing temperature and extension time at 63° C. for 3 min.Primers and probes concentrations were: BAT-26: 0.2×; ACVR2A: 0.6×;DEFB105A/B: 1×.

RESULTS

Materials and Methods

Primers and Probe Design

Primers and probes were designed with the support of Primer3PlusSoftware (Whitehead Institute for Biomedical Research). All primers werechecked for non-specific binding using Primer BLAST and absence ofsecondary structures. Primers were designed to generate ampliconssmaller than 140 bp for optimal amplification of cell free DNA (cfDNA)and fragmented DNA extracted from formalin-fixed paraffin-embedded(FFPE) tumor samples. Oligonucleotide sequences used in this study areprovided in Table 1. BAT-26 singleplex: SEQ IDs. 1-4; ACVR2A singleplex:SEQ IDs. 5-8; DEFB105A/B singleplex: SEQ IDs. 9-12; BRAF V600Esingleplex: SEQ ID. 13-16; BAT-26-ACVR2A-DEFB105A/B triplex: SEQ IDs.1-5, 7, 9, 11, 17-20. Desalted primers and HPLC-purified probes weremanufactured by Invitrogen and Applied Biosystems UK.

ddPCR Conditions

Droplet digital PCR (ddPCR) was performed using the Bio-Rad QX100 systemas instructed by the manufacturer. PCR reactions were prepared in a 20μL volume containing 10 μL of 2× Supermix for Probes without dUTP(Bio-Rad ref. 1863024), 900 nM of each primer, 250 nM of each TaqMan®probe and up to 16.5 ng of DNA template, which is equivalent to 5,000copies. The PCR reaction was then transferred to a disposable dropletgenerator cassette (Bio-Rad ref. 864008). 70 μL of droplet generationoil (Bio-Rad ref. 1863005) was added and the cassette loaded into thedroplet generator. Generated droplets (40 μL) were transferred to a96-well PCR plate (Eppendorf ref. 0030 128.575). Emulsified PCRreactions were then run on a C1000 thermal cycler (Bio-Rad) under thefollowing cycling conditions: denaturation at 95° C. for 10 min followedby 40 amplification cycles of 94° C. for 30 sec, 61° C. for 3 min(BAT-26) or 59° C. for 3 min (DEFB105A/B) or 55° C. for 3 min (ACVR2A)or 60° C. for 1 min (BRAFV600E); final hold at 98° C. for 10 min. Ramprate was set to 2.5° C./sec. At each run, controls with no DNA andcontrols containing 100% WT or 100% mutant DNA were included. Clusterthresholding and quantification was performed with the QuantaSoft v1.7.4software (Bio-RAD). For the ddPCR MSI assays, droplets were manuallyassigned as WT or MSI positive based on their fluorescence amplitude:WT, VIC⁺/FAM⁺, MSI positive (mutant), VIC⁺/FAM^(−/low). Droplets with notemplate were assigned VIC⁻/FAM⁻. Assay optimization was performed withgenomic DNA (gDNA) of HCT-116 cell line (a MSI positive colon cancercell line) diluted or not in WT DNA obtained from peripheral bloodmononuclear cells (PBMC). From droplets counts through manualassignment, mutant allele frequencies (MAF) were determined.

LOB and LOD Calculations

The background signal or false-positive rate of each assay was estimatedusing at least 53 replicates of WT DNA. The limit of blank (LOB) wasdefined as the upper 95% confidence limit of the mean false-positivemeasurements. The analytical sensitivity was estimated using serialdilutions of HCT-116 cell line in WT DNA, in mutant allele frequencies(MAF) ranging from 10% to 0.01% (1:2 serial dilutions). The total numberof replicates per dilution point ranged from 3 to 8 (10% and 5%, 3×;2.5% and 1.25%, 4×; 0.63% to 0.16%, 6×; 0.08% to 0.01%, 8×) in order tomaximize the detection of rare events. The limit of detection (LOD) wasestimated as the lowest mutant concentration likely to be reliablydistinguished from the LOB.

Validation of the ddPCR MSI Assays in Patient Samples

Formalin-fixed paraffin-embedded (FFPE) tumor tissue, plasma or serumsamples of patients with predominantly colorectal cancer (CRC) orendometrial carcinomas (EC) were used to validate the ddPCR MSI assays.All samples were obtained from patients treated and enrolled in clinicalstudies at the Institut Curie (Paris, France), with approval from theInstitution's Clinical Research Ethical Board. Samples were selectedfrom a pool of microsatellite stable (MSS) or microsatellite instable(MSI-H) tumors, identified by the pentaplex PCR method (Bacher et al2004) in association or not with immunohistochemistry staining (IHC) ofmismatch repair (MMR) proteins (MLH1, MSH2, MHS6 and PMS2). gDNA fromtumor tissues was extracted using the Qiagen DNA FFPE Tissue Kit (Qiagenref. 56404) according to the manufacturer's instructions and stored at−20° C. cfDNA was extracted from 0.5 to 1.8 mL of plasma or serum usingthe QIAamp® Circulating Nucleic Acid Kit (Qiagen ref. 55114), followingthe manufacturer's recommendations and stored at −20° C. DNA wasquantified using Qubit dsDNA HS assay and LINE-1 amplification (Rago etal 2007). ddPCR reactions were performed as described above. Total DNAamount per reaction varied from 2.5 ng to 10 ng for FFPE samples andfrom 1 ng to 10 ng for plasma or serum samples.

Results

BAT-26, ACVR2A and DEFB105A/B MSI ddPCR Assays Reliably Detect AlleleSize Variations in the Microsatellites Located Inside MSH2, ACVR2A andDEFB105A and B Genes, Respectively

We developed ddPCR assays capable of detecting allele size variationsfor 3 mononucleotide poly(A) microsatellite (MS) markers: BAT-26, aquasi-monomorphic long A₂₇ repeat located at the fifth intron of MSH2gene, and two shorter A₈ and A₉ repeats located in the tenth exon ofACVR2A and second intron of DEFB105A/B paralogous genes, respectively(Table 1). BAT-26 is one of the five microsatellite markers widely usedto determine the MSI status of colorectal and endometrial tumors inclinical practice (Suraweera et al 2002). The microsatellites locatedwithin ACVR2A and DEFB105A/B genes are novel discriminatory markersrecently identified from the analysis of TOGA exome sequencing data asrecurrently unstable in MSI-H tumors, as compared to MSS tumors (Hauseet al 2016; Maruvka et al 2017). The three assays are based on thedrop-off ddPCR strategy, which identifies mutated alleles based on theabsence of a WT signal (Decraene et al 2018). For each microsatellitemarker two Taqman hydrolysis probes were designed within the sameamplicon. A VIC labelled reference probe (REF), which hybridizes to anon-variable sequence upstream or downstream of the microsatelliteregion and a FAM labelled drop-off probe (MS), which covers the entirepoly-A homopolymer plus 2 to 4 bases on either side to confer itsability to bind properly and the resulting destabilization in case ofmutated alleles associated with microsatellite instability. While theREF probe quantifies the total number of copies of the amplicon (i.e.BAT-26, ACVR2A or DEFB105A/B DNA fragments), the MS probe discriminatesWT and MSI alleles due to inefficient hybridization to mutant sequences.Therefore, with this type of assay 2-D scatter plots of VIC and FAMfluorescence amplitude may show three possible clusters of droplets:droplets with no template (VIC⁻/FAM⁻), droplets containing WT alleles(VIC⁺/FAM⁺) and droplets containing MSI positive alleles(VIC⁺/FAM^(−/low)) (FIGS. 1A to 1I).

Given the low complexity of the MS probe, adjustments to standard ddPCRconditions (BioRAD guidelines) had to be made in order to achievespecific hybridization to WT alleles. We observed that a thermal cyclingprotocol with increased annealing temperature and annealing/extensiontime improved significantly the specificity of the MS probe to WTalleles and, accordingly, improved the separation of the WT andMSI-positive clouds. Optimized assays were able to specifically detectMSI alleles in DNA extracted from HCT-116 MSI-H cell line while noinstability could be observed in WT DNA derived from peripheral bloodmononuclear cells (PBMC) (FIGS. 1A and 1B for BAT-26; 1D and 1E forDEFB105A/B; 1G and 1H for ACVR2A). Moreover, the three assays were ableto accurately quantify MSI alleles in 1/10 dilutions of HCT-116 cellline in a WT background (FIGS. 10, 1F, 1I).

BAT-26, ACVR2A and DEFB105A/B ddPCR Assays are Highly Specific and Reacha Limit of Detection Below 0.1%

Analytical specificity of BAT-26, ACVR2A and DEFB105A/B ddPCR MSI assayswas evaluated by measuring false-positive MSI calls in at least 53individual ddPCR reactions of WT DNA derived from PBMCs (average numberof copies per reaction: 4520 for BAT-26; 3380 for ACVR2A and 3740 forDEFB105A/B). Mean false positive rates were: 0.006908±0.01366% forBAT-26 (MSI calls in 11/53 reactions), 0.006136±0.01623% for ACVR2A (MSIcalls in 7/55 reactions) and 0.005604±0.01911% for DEFB105A/B (MSI callsin 5/55 reactions). The limit of blank (LOB) of each assay was estimatedat 0.01067% for BAT-26 (FIG. 2A), 0.01077% for DEFB105A/B (FIG. 2B) and0.01052% for ACVR2A (FIG. 2C). The analytical sensitivity was estimatedusing serial dilutions of HCT-116 cell line in WT PBMC DNA, in mutantallele frequencies (MAF) ranging from 10% to 0.01% (1:2 serialdilutions). The total number of replicates per dilution point rangedfrom 3 to 8 (10% and 5%, 3×; 2.5% and 1.25%, 4×; 0.63% to 0.16%, 6×;0.08% to 0.01%, 8×) in order to maximize the detection of rare events.For the three assays, excellent linear correlations were observedbetween the expected and observed MAF, indicating that the three assayscan accurately quantify MSI in a wide range of frequencies, R²=0.9984p<0.0001 for BAT-26 (FIG. 2A), R²=0.9964 p<0.0001 for DEFB105A/B (FIG.2B) and R²=0.9955 p<0.0001 for ACVR2A (FIG. 2C). The limit of detection(LOD), estimated as the lowest mutant concentration likely to beaccurately distinguished from the LOB was estimated at 0.04% for BAT-26(FIG. 2A) and 0.08% for both DEFB105A/B (FIG. 2B) and ACVR2A markers(FIG. 2C). We conclude that the three MSI ddPCR assays are both highlysensitive and specific, promising better diagnostic accuracy and theunprecedented use of a MSI biomarker in liquid biopsies for diagnosisand monitoring of disease treatment and progression.

ddPCR MSI Testing in Clinical Samples

We next evaluated the performance of the BAT-26, ACVR2A and DEFB105A/BddPCR MSI assays in 177 FFPE tumor samples obtained predominantly frompatients with colorectal or endometrial cancers (Table 2). These sampleshad been previously characterized as MSI positive (MSI-H, n=94) or MSInegative (MSS, n=83) using the standard multiplex-PCR capillaryelectrophoresis method which evaluates microsatellite instability in 5microsatellite markers: BAT-26, NR-21, BAT-25, MONO-27 and NR-24.Samples showing instability for at least 2 of the 5 markers wereconsidered MSI positive (MSI-H), while samples showing no instabilitywere classified as MSI negative (MSS). Importantly, ddPCR and followinganalyses were performed blindly, without knowledge of the MSI status ofsamples. As shown in Table 2, MSI ddPCR identified unstable alleles forBAT-26, ACVR2A and DEFB105A/B markers in 92, 87 and 81 samples,respectively. Noteworthy for BAT-26 concordant results between capillaryelectrophoresis and ddPCR were obtained for 172 out of the 177 samplestested. For 3 of the 5 discordant samples, BAT-26 status could not bedetermined by capillary electrophoresis, but was defined as unstable byddPCR. For the other 2 discordant samples, BAT-26 was classified asunstable by capillary electrophoresis but was reported as stable andundetermined by ddPCR. Considering a sample as MSI-H if instability wasfound for at least 2 out of the 3 ddPCR markers analyzed, MSI ddPCRcould correctly classify 100% (83/83) of the MSS samples as MSS and 94%( 88/94) of the MSI-H samples as MSI-H. Of note, most of the discordantcases corresponded to endometrial tumor samples ( 4/6) which are moredifficult to classify than colorectal cancers and more prone forfalse-negative results (Suraweera et al 2002; Wang et al 2017).

Given the high sensitivity and specificity of the MSI ddPCR assays, wenext evaluated their performance on 22 plasma or serum samples collectedfrom 12 patients with stage IV MSI-H colorectal or endometrial tumors.Notable MSI ddPCR assays were able to detect microsatellite instabilityin all the samples tested, including samples with low mutant allelefrequencies, close to 0.2% (Table 3). Moreover, five of these 12patients had BRAF mutated tumors (BRAF V600E). Therefore, mutant allelefrequencies reported by the MSI ddPCR assays could be directly comparedwith the ones obtained with a ddPCR assay that targets specifically BRAFV600E mutation. Excellent correlations were obtained (R²=0.9852 p<0.0001for BAT-26, R²=0.9603 p<0.0001 for ACVR2A and R²=0.9275 p<0.0001 forDEFB105A/B), which further supports the reliability of the ddPCR MSIassays for detection and quantification of circulating tumor DNA (FIGS.3A to 3C). Taken together, these results demonstrate that the MSI ddPCRassays can accurately detect MSI in patient samples and therefore, canbe used as an alternative method for MSI testing in tumor tissue andliquid biopsies in clinical practice.

Development of a Multiplex Assay

We next aimed at developing a multiplex MSI ddPCR assay that cansimultaneously detect MSI status for BAT-26, ACVR2A and DEFB105A/Bmarkers in a single reaction. The multiplex strategy consisted invarying the concentrations of primers and probes in order to changeend-point fluorescence so that WT and MSI-positive clusters of dropletsfor the 3 markers could be distinguished from each other (see Bio-Raddroplet digital PCR multiplexing guideline). Different primers andprobes as well as diverse combinations of primer and probeconcentration, annealing temperature and extension time were tested,some of which generated satisfactory results. One example, obtained withannealing/extension temperature/time at 63° C. for 3 min and thefollowing primer/probe combinations: BAT-26, SEQ IDs. 1-4, 0.2×, ACVR2A,SEQ IDs. 5, 7, 17 and 18, 0.6× and DEFB105A/B, SEQ IDs. 9, 11, 19 and20, 1× is presented in FIG. 4 . These results, although preliminary,demonstrate the feasibility of multiplexing ddPCR assays targetingdiverse microsatellite sequences in a single reaction.

TABLE 1 List of primers and probes BAT-26 Primer Fw SEQ ID NO. 1GACTTCAGCCAGTATATGAAATTGGATATTG BAT-26 Primer Rev SEQ ID NO. 2GTATATGTCAATGAAAACATTTTTTAACCATTCAAC BAT-26 Probe REF SEQ ID NO. 3VIC-AGCAGTCAGAGCCCTTAACCTTT-MGB-NFQ BAT-26 Probe MS SEQ ID NO. 4 FAM-AGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAGG- MGB-NFQ ACVR2A Primer Fw SEQ ID NO. 5GAGGAGGAAATTGGCCAGCATC ACVR2A Primer Rv SEQ ID NO. 6AGCTAACTGGATAACTTACAGCATG ACVR2A Probe REF SEQ ID NO. 7VIC-ACTTCCTGCATGTCTTCAAGAG-MGB-NFQ ACVR2A Probe MS SEQ ID NO. 8FAM-CCTCTTTTTTTTATGC-MGB-NFQ DEFB105A/B Primer Fw SEQ ID NO. 9TTGAAAAATCTGGGCTGATTCTTGA DEFB105A/B Primer Rev SEQ ID NO. 10TGAGGGAGCTTTCCAGGAAATG DEFB105A/B Probe REF SEQ ID NO. 11VIC-CTTTGACATGTTCCCCATTTCTAG-MGB-NFQ DEFB105A/B Probe MS SEQ ID NO. 12FAM-TCCCTTTTTTTTTGGT-MGB-NFQ BRAF Primer Fw SEQ ID NO. 13TGAAGACCTCACAGTAAAAATAGGTGA BRAF Primer Fw SEQ ID NO. 14ACTGATGGGACCCACTCCATC BRAF Probe WT SEQ ID NO. 15VIC-TAGCTACAGTGAAAT-MGB-NFQ BRAF Probe V600E SEQ ID NO. 16FAM-CTAGCTACAGAGAAAT-MGB-NFQ ACVR2A Primer Rv-1 SEQ ID NO. 17CAGCATGTTTCTGCCAATAATCTC ACVR2A Probe MS-1 SEQ ID NO. 18FAM-AGGCCTCTTTTTTTTATG-MGB-NFQ DEFB105A/B Primer Rev-1 SEQ ID NO. 19GCCAAGAAAGAGCTGCTGAG DEFB105A/B Probe MS-1 SEQ ID NO. 20FAM-AACTGTCCCTTTTTTTTTGGT-MGB-NFQ

TABLE 2 Instability patterns obtained by pentaplex-PCR (*BAT-26, NR-21,BAT-25, Mono-27 and NR-24) and ddPCR MSI assays in FFPE tumor samples.Pentaplex ddPCR Tumor Profile* Classification BAT-26 ACVR2A DEFB105Classification colon ND++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + − MSI-H colon +++++ MSI-H + − + MSI-H colon+++++ MSI-H + − + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++MSI-H + − + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon+++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon ND++++ MSI-H + + + MSI-H colon+++++ MSI-H + + + MSI-H colon ++−++ MSI-H + + + MSI-H colon +++++MSI-H + + + MSI-H colon +++−− MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon++++− MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon+++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++MSI-H + + + MSI-H colon ++++− MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon+++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon+++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon+++++ MSI-H + + + MSI-H colon +−+++ MSI-H + + + MSI-H colon +++ ++MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon+++++ MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++MSI-H + + + MSI-H colon +++++ MSI-H + + + MSI-H colon +++++ MSI-H + + +MSI-H colon +++++ MSI-H + + + MSI-H endometrial +−−+− MSI-H + + − MSI-Hendometrial +++++ MSI-H + + + MSI-H endometrial ND++++ MSI-H ND + +MSI-H endometrial +++++ MSI-H + + + MSI-H endometrial +−−++ MSI-H + + −MSI-H endometrial ND++++ MSI-H + + − MSI-H endometrial +++++ MSI-H + − +MSI-H endometrial +−+−+ MSI-H + + − MSI-H endometrial +++++ MSI-H + + −MSI-H endometrial +++++ MSI-H + + − MSI-H endometrial +−+−+ MSI-H + + +MSI-H endometrial +++++ MSI-H + + + MSI-H endometrial +++++ MSI-H + + +MSI-H endometrial +++++ MSI-H + + + MSI-H endometrial +++++ MSI-H + + +MSI-H endometrial +++++ MSI-H + + + MSI-H cholangiocarcinoma +++++MSI-H + + + MSI-H intestine +++++ MSI-H + + + MSI-H rectum +++++MSI-H + + + MSI-H rectum +++++ MSI-H + + + MSI-H sebaceome +−++−MSI-H + + + MSI-H stomach +++++ MSI-H + + + MSI-H colon ND+ND+− MSI-H ND− − MSS ovary ND−+++ MSI-H ND + − MSS endometrial +++++ MSI-H ND + − MSSendometrial +++++ MSI-H + − − MSS endometrial +−+−− MSI-H − − − MSSendometrial ++ + + + MSI-H + − − MSS colon −−−−− MSS − − − MSS colon−−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSScolon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − −− MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−−MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon−−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSScolon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − −− MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−−MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon−−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSScolon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − −− MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−−MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon−−−−− MSS − − − MSS colon −−−−− MSS − − − MSS colon −−−−− MSS − − − MSScolon −−−−− MSS − − − MSS colon +−−−− MSS + − − MSS colon +−−−− MSS + −− MSS colon +−−−− MSS + − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − − − MSS endometrial −−−−− MSS − − − MSSendometrial −−−−− MSS − + − MSS ovary −−−−− MSS − − − MSS ovary −−−−−MSS − − − MSS rectum −−−−− MSS − − − MSS rectum −−−−− MSS − − − MSSrectum −−−−− MSS − − − MSS rectum −−−−− MSS − − − MSS rectum −−−−− MSS −− − MSS rectum −−−−− MSS − − − MSS rectum −−−−− MSS − − − MSS rectum−−−−− MSS − − − MSS rectum −−−−− MSS − − − MSS pancreas −−−−− MSS − − −MSS pancreas −−−−− MSS − − − MSS rectum −−−−− MSS − − − MSS ND: nondetermined

TABLE 3 Mutant allele frequencies obtained by ddPCR MSI assays in bodyfluid samples collected from patients with stage IV MSI-H colorectal orendometrial tumors. Patients with BRAF^(V600E) mutated tumors are markedby an asterisk MSI-ddPCR MAF (%) Patient Primary tumor Sampling SampleBAT-26 DEFB106 ACVR2A before plasma 25.74 19.80 22.30 treatment P-01*colon progression plasma 0.35 0.32 0.43 P-02 colon pre-surgery serum0.52 0.62 0.66 pre-surgery serum 0.23 — — P-03 colon 1^(st) serum 4.202.88 — progression 2^(nd) serum — 0.21 — progression before plasma 62.0050.88  53.10  treatment P-04* colon treatment plasma 0.45 0.39 0.28treatment plasma 2.80 2.10 2.60 progression plasma 1.70 — 2.90 treatmentplasma 2.09 1.40 1.90 P-05* colon treatment plasma 6.80 4.07 5.30treatment plasma 0.26 — 0.23 P-06* colon treatment plasma 11.40 — 6.60treatment plasma 13.10 — 5.50 P-07 colon before plasma 45.27 13.52 37.90  treatment P-08* endometrium treatment plasma 1.82 0.24 1.47 P-09endometrium pre-surgery serum 5.70 1.05 1.60 P-10 endometriumpre-surgery plasma 0.65 — P-11 endometrium pre-surgery serum 0.25 — P-12endometrium pre-surgery serum 1.32 — pre-surgery serum — 0.31 —

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1. A method for detecting a mutation in a microsatellite sequence locusof a target fragment from a DNA sample, comprising a step of subjectingsaid DNA sample to a digital polymerase chain reaction (dPCR) in thepresence of a PCR solution comprising: a pair of primers suitable foramplifying said target fragment of the DNA sample including saidmicrosatellite sequence; a first MS oligonucleotide (MS) hydrolysisprobe, labeled with a first fluorophore, wherein said first MSoligonucleotide probe is complementary to a wild-type sequence includingthe microsatellite sequence; a second oligonucleotide reference (REF)hydrolysis probe, labeled with a second fluorophore, wherein said secondoligonucleotide REF probe is complementary to a wild-type sequence ofsaid target DNA fragment located outside of said microsatellitesequence.
 2. The method according to claim 1, wherein the targetfragment of the DNA sample is constitutional genomic DNA.
 3. The methodaccording to claim 1, wherein the target fragment of the DNA sample isgenomic tumor DNA.
 4. The method according to claim 1, wherein themicrosatellite sequence locus is selected from the group comprisingBAT-25, BAT-26, BAT-34c4, BAT-40, NR21, NR24, MONO-27, D2S123, D5S346,D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3, GTF2IP1,LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1 and TDRD1.
 5. The methodaccording to claim 1, wherein the DNA sample is selected from the groupconsisting of tumor tissue, disseminated cells, feces, blood cells,blood plasma, serum, lymph nodes, urine, saliva, semen, stool, sputum,cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice,amniotic fluid, cerebrospinal fluid, serous fluids.
 6. The methodaccording to claim 1 further comprising a step of measuring thefluorescence signals associated with the REF and MS probes, wherein themaximal fluorescence intensity signal associated with both the REF andMS probes indicates the presence of a wild-type microsatellite sequencein the target DNA fragment, while a shift in the fluorescence intensitysignal associated with the MS probe indicates the presence of a mutationin the microsatellite sequence of the target DNA fragment
 7. A methodfor the detecting cancers, diseases associated with mutations inmismatch repair (MMR) genes or familial tumor predisposition in asubject, comprising the detection of a mutation in a microsatellitesequence locus of a target DNA from a DNA sample according to claim 1,wherein the target fragment is originating from a tumor.
 8. A method forprognosis of cancers comprising the detection of a mutation in amicrosatellite sequence locus of a target fragment from a DNA sampleaccording to claim 1, wherein the target fragment is originating from atumor.
 9. A method for predicting the efficacy of a treatment in asubject suffering from a cancer, comprising the detection of a mutationin a microsatellite sequence locus of a target fragment from a DNAsample according to claim 1, wherein the target fragment is originatingfrom a tumor and wherein the treatment is preferably immune therapy suchas immune checkpoint therapy.
 10. A method of treatment of a cancer in asubject in need thereof comprising: the detection of a mutation in amicrosatellite sequence locus of a target fragment from a DNA sampleaccording to claim 1, and the administration to the subject of animmunotherapy if a mutation is identified in a microsatellite sequencelocus of the target fragment, wherein the target fragment of the DNAsample originates from a tumor.
 11. A method for the monitoring of apatient diagnosed with a tumor associated with impaired DNA mismatchrepair (MMR), or having suffered from such tumor, comprising thedetection of a mutation in a microsatellite sequence locus of a targetfragment from a DNA sample, wherein the target fragment of the DNAsample originates from a tumor.
 12. A kit for identifying a mutation ina microsatellite sequence region of a target fragment from a DNA samplecomprising: a pair of primers suitable for amplifying said targetfragment from the DNA sample including said microsatellite sequence; afirst oligonucleotide hydrolysis probe (MS), labeled with a firstfluorophore, wherein said first oligonucleotide probe is complementaryto a wild-type sequence including the microsatellite sequence; a secondoligonucleotide hydrolysis probe (REF), labeled with a secondfluorophore, wherein said second oligonucleotide probe is complementaryto a wild-type sequence of said amplified DNA fragment located outsideof said microsatellite sequence; and a thermostable polymerase.